A novel CCR5 inhibitor, GRL-117C, we designed and synthesized turned out to exert potent activity against R5-HIV-1Ba-L with a sub-nanomolar IC50 value in the MAGI assay using MAGI/CCR5 cells. The potency (IC50 values) of GRL-117C was comparable to that of MVC, as was determined by both the MAGI assay (0.6 nM vs. 0.7 nM) and the p24 assay with PBMCs (8.1 nM vs. 4.5 nM). The other GRL-compounds, GRL-10007C and GRL-10018C, also demonstrated strong activity against HIV-1Ba-L in the MAGI assay (IC50: 1.4 nM and 2.9 nM, respectively). Two drug-naive clinical R5-HIV-1 strains, CC1/85 cl.6 and cl.7, were also used in the assays. All the compounds tested in this study showed similar effectiveness against the CC1/85 clinical strains compared to HIV-1Ba-L. The IC50 value of GRL-117C for the MAGI assay was 0.6 nM, but was 8.1 nM for the p24 assay (HIV-1Ba-L). Activity of CCR5 inhibitors against transmitter/founder (T/F) HIV-1s. We also examined the activity of GRL-compounds against Transmitter/founder (T/F) HIV-1 viruses. T/F viruses are involved in the initial infection. It is considered that T/F viruses virtually always use CCR5 rather than CXCR4 and infect T cells but not macrophages because high level of CD4 is needed to mediate virus entry for the initial transmission. Thus, CCR5 inhibitors are expected to be active against such T/F viruses. We obtained four HIV-1 T/F infectious clones. We determined the antiviral activity of GRL-117C, GRL-10007C and GRL-10018C on these HIV-1 clones using MAGI assay. We also determined the activity of Maraviroc and APL against these viruses. GRL-CCR5 inhibitors, especially GRL-117C, exerted potent activity against all four T/F viruses. Maraviroc and APL were also highly potent. The IC50 values of GRL-117C were 1.9-3.2 nM, and were substantially similar to the activity of MVC (1.7-2.3 nM) against these cells. Activity of CCR5 inhibitors against CCR5 inhibitor-resistant HIV-1s. We selected three compounds (GRL-117C, GRL-10007C, and GRL-10018C) for further testing. In a previous study, Trkola et al., reported the generation of an escape mutant HIV-1 for AD101 (experimental CCR5 inhibitor), and found that this mutant did not use CXCR4, but instead gained the ability to use CCR5 in an AD101-insensitive manner. Subsequently, Marozsan et al., described the generation of escape mutants under the selection pressure of VVC in vitro. Both escape mutants were fully resistant against AD101 and VVC. For the current study, AD101- and VVC-resistant HIV clones were provided by Dr. John P. Moore of Cornell University. CC101.19 (AD101-resistant) was approximately 150-fold more resistant to SCH-C (IC50: 1,000 nM) compared to its corresponding CCR5 inhibitor-sensitive viruses, CC1/85 (cl.6 and cl7, IC50: 5.2 and 6.1 nM, respectively). On the other hand, resistance against other CCR5 inhibitors, including MVC, APL, and GRL-compounds, were relatively lower in comparison; fold resistance ranged from 2.6- to 15-fold. The VVC- resistant virus (D1/85.16) also showed high resistance against SCH-C (68-fold), but remained susceptible to all other drugs to some extent (fold resistance: 3.6-fold to 12.5-fold). GRL- 117C exhibited slightly decreased activity against AD101- and VVC-resistant viruses (fold resistance: 9.3- and 8.5-fold, respectively), however, its IC50 numbers remained less than 40 nM. Interestingly, GRL-10007C, which was less reactive than GRL-117C against wild type R5-HIV-1, maintained its activity against AD101- and VVC-resistant viruses, showing IC50 values of 41.1 nM (2.6-fold) and 56.9 nM (3.6-fold). This result suggested that the resistance profiles of SCH-C and its associated drugs (VVC and AD101) differ drastically from those of MVC, APL, and GRL-derivatives. GRL-10007C, which induced the least resistance in these viruses, may have a unique resistance profile among the CCR5 inhibitors tested in this study. We then wanted to determine if these compounds are effective against HIV-1s carrying MVC-resistance-associated substitutions. The activity of GRL-117C was reduced when used against the highly MVC-resistant virus (HIV-1KP-5mvcR) (IC50: 686 nM). However, GRL-117C also demonstrated decreased activity against a drug- naive HIV-1 clinical strain (HIV-1KP-5pc) as compared to the laboratory HIV-1 strain (HIV- 1YU2). Therefore, while the fold change of IC50 values for GRL-117C was only 4.8 between HIV-1KP-5pc and HIV-1KP-5mvcR, we concluded that GRL-117C had cross-resistance with MVC because its IC50 value against HIV-1KP-5mvcR (686 nM) was more than 10-fold greater than that of MVC (41 nM). The other derivatives, GRL-10007C and GRL- 10018C also failed to demonstrate activity against HIV-1KP-5mvcR (data not shown). It is of note that the activity of CVC against HIV-1KP-5mvcR was substantially decreased compared to that of wild type [IC50: 260 nM vs. 4.1 nM (63-fold)], indicating that it also has cross- resistance with MVC. GRL-CCR5 inhibitors inhibit the binding of CC-chemokines to CCR5. In order to determine whether GRL derivatives block the binding of CC-chemokines to CCR5, we conducted a CC-chemokine binding inhibition assay using 125I-labeled CC-chemokines and CCR5 expressing cells. All the CCR5 inhibitors tested (GRL-117C, GRL-10007C, GRL-10018C, MVC, and APL) blocked the binding of 125I MIP to CCR5, and their EC50 values ranged from 0.1-4.3 nM. Similar results were observed for MIP-1?binding (EC50 range: 0.2-2.5 nM). Results demonstrated that MVC, APL, and GRL-10018C exert stronger inhibitory effects on MIP-1??and MIP-1?binding compared to GRL-117C and GRL-10007C. In contrast, APL, GRL- 117C, and GRL-10007C only moderately blocked the binding of RANTES; their EC50 values were 156, 121, and 628 nM, respectively, and binding of 125I-RANTES remained at more than 40%. Three-dimensional models of human CCR5-CCR5 inhibitor complexes were defined using the crystal structure of CCR5-MVC as the template (PDB ID: 4MBS). MVC was found to be lodged in the bottom of the largest pocket at the binding site, which was defined by residues from helices 1, 2, 3, 5, 6, and 7. It was observed that MVC forms hydrogen bonds with Tyr-37, Tyr-251, and Glu-283, and its phenyl group reaches deep into the pocket to form hydrophobic interactions with aromatic residues such as Tyr-108, Trp-248, and Tyr-251. GRL-117C also binds to the same binding cavity, and similar to MVC, GRL-117C forms hydrogen bonds with Tyr-37 and Glu-283, but not with Tyr-251. Binding models of GRL-10018C with CCR5 also showed formation of hydrogen bonds between GRL-10018C and Tyr-37 and Glu-283. On the other hand, overall, APL binds in the same active site cavity as previously reported. There are polar interactions with Tyr-37, Ser-180 and Thr-195. Tyr-108, Tyr-251 and Glu-283 are part of the binding pocket and form non-polar interactions with APL. As described, GRL compounds (117C and 10018C) and MVC have hydrogen-bond interactions with Tyr-37 and Glu-283. MVC also has a hydrogen-bonding with Tyr-251. On the other hand, for GRL-117C and 10018C, there are weak non-polar interactions and very weak pi-pi interactions between the phenyl groups of GRL-compounds and Tyr-251, thus making slightly different binding profiles of GRL-compounds to the CCR5 binding cavity comparing to that of MVC. As was also described in the previous section, GRL-10018C exhibited more potent inhibitory effects on the binding of the three chemokines as compared to GRL-117C and GRL-10007C. This may be due to the fact that GRL-10018C possesses a bis-THF structure. The bulky rings occupying the upper region of the binding cavity under ECL2 cause steric hindrance with CC-chemokine when it binds to CCR5. However, the bis-THF structure does not affect the interaction between gp120 with CCR5.